Radiated light filtering for a flow cytometer

ABSTRACT

A filter mask for use in a flow cytometer includes light blocking features and light passing apertures. The flow cytometer operates to evaluate one or more characteristics of a sample by illuminating the sample and a carrier fluid and collecting light rays that are radiated from the sample and the carrier fluid. The light rays are passed through the filter mask. The light blocking features of the filter mask are arranged to selectively block radiated light at certain radiation angles, while permit light rays having other radiation angles to pass therethrough. A sensor analyzer receives the light rays that pass through to evaluate at least one characteristic of the sample. The light rays can also be separated into two beams, which can be independently filtered using different filter masks. The results can then be compared to provide even more information regarding characteristics of the sample.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a National Stage of PCT International Patentapplication No. PCT/US2014/029058, filed 14 Mar. 2014, which claimsbenefit of U.S. Ser. No. 61/798,548, filed on 15 Mar. 2013, titledRADIATED LIGHT FILTERING FOR A FLOW CYTOMETER and which applications areincorporated herein by reference in their entireties. To the extentappropriate, a claim of priority is made to each of the above disclosedapplications.

BACKGROUND

Flow cytometers are used to evaluate the content of a sample. The sampleis introduced into a fluid stream, which is then illuminated with alight beam. When the light beam enters the fluid, it interacts with thesample and light is radiated and fluoresced out from the fluid invarious directions. By evaluating the way that the light radiates fromthe fluid, characteristics of the sample can be determined.

SUMMARY

In general terms, this disclosure is directed to radiated lightfiltering for a flow cytometer. In one possible configuration and bynon-limiting example, a filter mask is provided which selectivelyfilters light radiated by a sample.

One aspect is a filter mask for use in a flow cytometer, the filter maskcomprising: a body including: light blocking features including: anouter blocker; and a secondary blocker configured to block light rayshaving blocked radiation angles; and light passing apertures configuredto permit light rays having radiation angles greater than and less thanthe blocked radiation angles to pass through the body.

Another aspect is a flow cytometer comprising: a flow nozzle configuredto provide a fluid along a flow path, the fluid including sampleparticles therein; a light source configured to generate a light beamdirected toward the flow path, wherein when the light beam intersectswith the flow path, light rays are radiated by the fluid and theparticles at radiation angles; an optics system configured to receivethe radiated light rays and to direct the light rays along an opticalpath, the optics assembly including at least a first filter mask, thefirst filter mask including light blocking features positioned in thefirst filter mask to selectively block light rays having specificradiation angles; and a sensor analyzer arranged at an end of theoptical path to collect and analyze light rays passing through theoptics system.

A further aspect is a method of evaluating a particle with a flowcytometer, the method comprising: passing a particle in a fluid along afluid flow path; illuminating the particle and the fluid with a lightbeam; collecting light rays radiated from the fluid and the light beamwith an optics system; selectively blocking some of the light rayshaving certain radiation angles with a filter mask; selectively passingothers of the light rays with the filter mask; and detecting light rayspassed by the filter mask with a sensor analyzer to evaluate at leastone characteristic of the particle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic block diagram of an example flow cytometeraccording of the present disclosure.

FIG. 2 is a schematic diagram illustrating a side view of a portion ofthe example flow cytometer shown in FIG. 1.

FIG. 3 is a front elevational view of an example filter mask.

FIG. 4 is a front elevational view of another example filter mask.

FIG. 5 is a front elevational view of another example filter mask.

FIG. 6 is a front elevational view of another example filter mask.

FIG. 7 is a front elevational view of another example filter mask.

FIG. 8 is a front elevational view of another example filter mask.

FIG. 9 is a front elevational view of another example filter mask.

FIG. 10 is a cross sectional side view of an example optics system.

FIG. 11 is a cross-sectional top view of the example optics system shownin FIG. 11.

FIG. 12 is a schematic diagram illustrating an exemplary responseprofile for an example smoothness mask.

FIG. 13 is a schematic diagram illustrating an exemplary responseprofile for an example separation mask.

FIG. 14 is a front perspective view of an example mask holder.

FIG. 15 is a rear perspective view of the example mask holder shown inFIG. 14.

FIG. 16 is a front elevational view of the example mask holder shown inFIG. 14.

DETAILED DESCRIPTION

Various embodiments will be described in detail with reference to thedrawings, wherein like reference numerals represent like parts andassemblies throughout the several views. Reference to variousembodiments does not limit the scope of the claims attached hereto.Additionally, any examples set forth in this specification are notintended to be limiting and merely set forth some of the many possibleembodiments for the appended claims.

FIG. 1 is a schematic block diagram of an example flow cytometer 100. Inthis example, the flow cytometer 100 includes a flow nozzle 102, a lightsource 104, a sample collector 106, an optics system 108, a sensoranalyzer 110, and a computing device and control electronics 112. Theoptics system 108 includes a filter mask 120.

The flow nozzle 102 receives a sample containing particles for analysisby the flow cytometer 100. The flow nozzle 102 has a small aperture thatpermits only one or a small number of particles to pass through at atime, such as to arrange the particles so that they pass through theflow nozzle 102 in single file, for example. Examples of flow nozzles102 include a flow cell and a jet-in-air nozzle. In some embodiments,the flow cell includes a transparent body including a microscopicallythin channel. The fluid stream containing the particles is directed bythe walls of the channel along the fluid path extending through the flowcell and past the light source 104. In other embodiments a jet-in-airnozzle is used to eject the fluid stream along the fluid path.Hydrodynamic forces cause the fluid to flow in a continuous fluid streamand confine the particles as they pass the light source 104. Otherembodiments utilize other flow nozzles 102.

The sample is mixed with a sheath fluid, and the resulting fluid steam Fcontaining the sample is directed along a flow path FP. The sample canbe of a variety of different types, and some embodiments will includemultiple types within a single sample. Examples of types of sampleparticles include beads, blood, bacteria, yeast, plankton,microparticles (e.g., from plasma membrane of cells), and mitochondria.

The light source 104 generates a light beam LB. An example of a lightsource 104 is a laser, which generates a laser beam. Other embodimentsuse other light sources, such as an arc lamp. The light beam LB isdirected to the fluid path FP in a direction A1 where the light beam LBenters the fluid. Although the light beam LB is typically directedtoward the fluid path FP by the light source 104 itself, the light beamLB can alternatively be directed by one or more optic devices, such aslenses, mirrors, prisms, and the like, in other embodiments after thelight beam is emitted from the light source 104.

The fluid stream F is directed to a sample collector 106 afterproceeding along the fluid path FP. In some embodiments, the samplecollector 106 is a waste receptacle. In other embodiments, the samplecollector 106 includes one or more storage receptacles. In anotherpossible embodiment, the flow cytometer 100 is a sorting flow cytometer,and the sample collector 106 operates to sort the particles in the fluidinto multiple receptacles based on one or more detected characteristicsof the particles.

When the light beam LB enters the fluid stream F, at least some of thelight rays LR are radiated (e.g., forward, side, or back) by theparticles within the fluid. Some of these light rays areforward-scattered, as shown in FIG. 1, while other light rays areside-scattered and back-scattered. Fluroescent light is also generated,which can also radiate in forward, side, or backward directions. Aradiation angle θ (sometimes also referred to as a scatter angle) is theangle of a light ray LR relative to the direction A1 of the light beamLB after being scattered or fluoresced by the fluid stream F. Becausethe light beam LB includes many light rays LR that can be separatelyradiated in different directions, different light rays LR can beradiated in different directions—having different radiation anglesθ—simultaneously. Forward-scattering (and fluorescence) is illustratedand described in more detail with reference to FIG. 2. Although FIG. 1illustrates only a vertical radiation angle θ, the light rays LR canalso be radiated in a horizontal dimension (i.e., in all threedimensions).

An optics system 108 is positioned adjacent the fluid path FP to receivethe radiated light rays. In some embodiments, the optics system 108includes a filter mask 120. The filter mask 120 is arranged andconfigured to block a portion of the light rays LR having certainradiation angles θ and to pass another portion of the light rays havingdifferent radiation angles θ. Examples of filter mask 120 are describedherein.

As discussed above, at least a portion of the optics system 108 istypically arranged adjacent the fluid path FP. In the illustratedexample, the optics system 108 is positioned a distance D1 away from thefluid path. Different embodiments can have different distances D1. Someembodiments have a distance in a range from about 10 mm to about 15 mm,for example.

After the light rays LR have passed through the optics system 108, theyare detected by the sensor analyzer 110. The sensor analyzer 110 detectsvarious characteristics of the light rays, such as one or more of themagnitude and position of the detected light, time duration of the lightpulse as a particle traverses the light beam, the shape of the pulse,polarization, and wavelength.

The computing device and control electronics 112 interact with thesensor analyzer 110 to evaluate characteristics of the particles in thefluid. In some embodiments, the computing device 112 includes a display,and generates a user interface on the display to convey informationregarding the characteristics of the particles in the fluid to a user.The computing device 112 typically includes at least one processingdevice (such as a central processing unit) and at least some form ofcomputer readable media, such as computer readable storage media.Examples of computer readable media are described herein.

In some embodiments, the flow cytometer 100 is a sorting flow cytometerin which the computing device and control electronics 112 operate tosort particles into multiple different receptacles in the samplecollector 106 based at least in part on the forward-radiated lightdetected by the sensor analyzer 110. For example, drops of the fluid areselectively charged by the flow nozzle 102 prior to separation from thefluid stream at the flow nozzle 102 based on detected characteristics ofthe particles contained in the drops. The drops are then sorted intodifferent receptacles by passing the drops through charged plates at thesample collector 106. The charged plates deflect the drops into theappropriate receptacles.

FIG. 2 is a schematic diagram illustrating a side view of a portion ofan example flow cytometer 100, such as a portion of the flow cytometer100 illustrated in FIG. 1. The illustrated portion of the flow cytometer100 depicts the light beam LB, fluid stream F, light rays LR radiated bythe fluid stream F, and the optics system 108. In this example, theoptics system 108 includes the filter mask 120.

When the light beam LB enters the fluid stream F, the fluid (and anyparticles contained in the fluid) cause the light rays LR to radiate indifferent directions (depicted by the radiation angles θ). The radiationoccurs both vertically and horizontally. The vertical radiation isillustrated in FIG. 2, which illustrates light rays LR being verticallyradiated between 45° (upward) and −45° (downward). The radiation alsooccurs horizontally, such as between −12° (left from the perspective ofthe light beam LB) and +12° (right). The radiation can also occuroutside of these ranges, and some embodiments collect, filter, and/orevaluate light rays LR outside of these ranges.

It has been found, however, that not all of the radiated light rays LRare equally informative when evaluating one or more characteristics of asample. Therefore, a filter mask 120 can be used to selectively blockcertain portions of the light rays, while permitting the light rays ofinterest to pass through.

As one hypothetical example, suppose that the only light rays ofinterest are those having a radiation angle θ from 20° to 35°, and from−20° to −35°. A filter mask 120 can be arranged and configured to blockthe undesired portion of the light rays (such as those having radiationangles between −20° and +20°, and those having radiation angles greaterthan +/−35°). The filter mask 120 can be similarly arranged andconfigured with apertures formed at precise locations that permit lightrays LR having radiation angles θ of interest (such as those havingradiation angles θ from 20° to 35°, and from −20° to −35°) to passthrough.

In some embodiments, the light rays LR are collected and redirectedalong an optical path by the optics system 108 before being filtered bythe filter mask 120. For example, in some embodiments the optics system108 includes one or more lenses which collect the light rays LR anddirect the light rays LR along the optical path.

Even though the directions of the light rays LR can change in the opticssystem, it is convenient to refer to the light rays by their radiationangles—the angles from which the light rays were radiated by the fluid.

Several exemplary light rays are shown in FIG. 2. Light rays that arenot radiated by the fluid continue along the axis A2 of the opticalpath, while light rays that were radiated are spaced from the axis A2.For example, some light rays may have a radiation angle of 15°, whileother light rays may be radiated at other angles, such as 30°, 45°,−15°, −30°, and −45° and other angles therebetween, and yet other anglesgreater than +/−45°. The light rays that have larger radiation anglesare spaced a greater distance away from the axis A2 than light rays thathave smaller radiation angles. In the illustrated example, the lightrays radiated at +/−15° radiation angles are spaced a distance +/−D2away from the axis A2 at a given point in the optics system 108 (such asa distance D1 from the fluid steam F). Similarly, the light raysradiated at +/−30° and +/−45° are spaced a distance +/−D3 and +/−D4,respectively from the axis A2. The distance D4 is greater than thedistance D3, which is greater than the distance D2. In some embodiments,the values of D2, D3, and D4 can be computed using basic trigonometryknowing the radiation angle of the light ray and the distance D1. Inother embodiments, a transfer function is used to map between theradiation angles and the positions of the light rays, as discussedherein. The distances D2, D3, and D4 can change as the light rays passthrough the optics system (such as caused by divergence or convergenceof the light rays by the lenses of the optics system), but the relativepositions of the light rays remain the same, in some embodiments.

As a result of this a filter mask 120 can be positioned in the opticssystem 108 to filter the light rays according to their radiationangles—to block certain light rays, while allowing other light rays topass therethrough. Examples of filter masks 120 are illustrated anddescribed with reference to FIGS. 3-9.

The light rays that pass through the filter mask 120 are then collectedby the optics system 108 and directed to the sensor analyzer 110, wherethe one or more characteristics of the sample are evaluated.

A benefit of the configuration shown in FIG. 2 is that precise filteringof light rays LR having particular radiation angles can be accomplished,limited primarily by the ability to precisely form apertures in thefilter mask 120, and the ability to properly position the filter mask120 with respect to the light beam LB direction A1.

Another benefit is that filter masks can be selected for use within theflow cytometer which have desired characteristics that are optimized fora particular application. For example, a first filter mask can beinserted into the optics system, which has a first set ofcharacteristics that make it useful for a first application. The filtermask can then be removed and replaced with a different filter mask for asecond application. In some embodiments, no changes need to be made tothe optics system 108, other than to remove the filter mask and replaceit with another filter mask having the desired characteristics.Additional examples are described herein.

FIG. 3 is a front elevational view of an example filter mask 120. Inthis example, the filter mask 120 includes a body 122, apertures 124,and an origin point 126.

The body 122 of the filter mask 120 is arranged and configured to blockcertain portions of the radiated light rays LR from passing. Forexample, in some embodiments the mask body 122 is formed of a materialthat will absorb most or all of the light rays LR. The filter mask 120can be formed of a material such as plastic or metal, for example. Inanother possible embodiment, the body is formed of one material and iscoated with one or more layers of material that absorb most or all ofthe light rays LR. For example, in some embodiments the body is formedof glass. A coating is applied to one surface of the glass, andphotolithography techniques are utilized to selectively remove thecoating to form apertures 124 through the coating. In this example, theapertures 124 do not extend entirely through the mask body 122, but onlythrough the coating, but due to the transparency of the glass, theapertures 124 still permit light rays to pass therethrough. Thephotolithography techniques can include, for example, masking of thelight-blocking portions of the body 122, and etching of the coating fromthe aperture 124 portions that are not protected by the masking layer.

The actual physical dimensions of the filter mask 120 are selecteddepending on the desired location of the filter mask within the opticssystem 108, and the known relative positions of the light rays havingvarious different radiation angles, as shown in FIG. 2 (e.g., distancesD2, D3, and D4). The overall height H1 and width W1 are selected to belarge enough to block the undesired light rays from proceeding along theoptical path.

In some embodiments, the filter mask 120 is positioned in the opticalpath at a location where the unradiated light rays (axis A2, shown inFIG. 2) are directed toward an origin point 126 of the filter mask 120.The origin point 126 is the point of the filter mask 120 at which theunradiated light rays are directed when the filter mask 120 is properlypositioned within the optics system 108.

The filter mask 120 shown in FIG. 3 includes apertures 124 (includingapertures 124A and 124B in this example) formed in the body 122. Theapertures 124 are positioned at particular locations in the body 122 topermit radiated light rays LR having certain radiation angles θ to passtherethrough. In this example, the apertures are positioned to permitlight rays LR having a vertical radiation angle between +/−20° and+/−35° and a horizontal radiation angle between −12° and +12° to passtherethrough. Accordingly, aperture 124A has a inner edge that is spaceda distance H2 from the origin point 126, corresponding to theanticipated position of light rays having a radiation angle of 20°. Anouter edge of the aperture 124A is spaced a distance H4 from the originpoint 126, corresponding to the position of light rays having aradiation angle of 35°. A left edge of the aperture 124A (from theperspective of the light beam) is positioned a distance W2 toward theleft of the origin point 126, and a right edge of the aperture 124A ispositioned at an equal distance W3 to the right of the origin point 126,corresponding to the horizontal radiation angles of +/−12°. The aperture124B is similarly positioned having an inner edge a distance H3 and anouter edge a distance H5 away from the origin point 126 in the opposite(i.e., negative) direction, and having the same left and right edgepositions.

In some embodiments, the specific positions of the filter mask 120apertures are determined using a transfer function. The transferfunction maps the radiation angles to the appropriate physical positionswithin the optics system. In some embodiments, the radiation angles arelinearly related to the physical positions of the filter mask features.In other embodiments, the transfer function may be non-linear, such ashaving a logarithmic, parabolic, or other non-linear relationship. Insuch embodiments, the transfer function can be determined according tothe specific characteristics of the optics system to permit mappingbetween the radiation angles and the physical positions for desiredfeatures of the filter masks.

FIG. 4 is a front elevational view of another example filter mask 120.In this example, the filter mask 120 includes a body 122 and apertures124. The body 122 includes four exemplary light blocking features,including an outer blocker 140, a secondary blocker 142, a centralblocker 144, and a bullseye blocker 146. Not all embodiments include allfour features. For example, some embodiments include one, two, or threeof these features.

As discussed herein, the body 122 is typically formed of a thin sheet ofone or more materials, such as plastic, metal, or glass. In someembodiments, the body 122 includes a coating of one or more layers ofone or more other materials. The body 122 operates to block light rayshaving certain radiation angles, while including apertures 124 thatpermit other light rays to pass therethrough. In some embodiments, theblocking portions of the body 122 are formed of a material thatsubstantially absorbs and/or blocks light rays, such as a materialhaving a dark color (e.g., black).

Apertures 124 are formed in the body 122 to permit light to passtherethrough. The apertures 124 can extend entirely through the body122, or through one or more layers of light absorbing and/or blockingmaterial. In some embodiments, the apertures 124 are transparent.

Each of the four exemplary features of the body 122 are described inturn below.

The outer blocker 140 is configured to block out light rays havingradiation angles greater than desired maximum vertical and horizontalradiation angles. In this example, the outer blocker 140 includes anouter edge 152 and an inner edge 154, and the outer blocker 140 extendstherebetween. The outer blocker 140 has a height and width (i.e.,similar to H1 and W1, shown in the example of FIG. 3) that are suitableto block light rays having radiation angles greater than the maximumvertical and horizontal radiation angles from proceeding along theoptical path. In some embodiments, the outer edge 152 of the outerblocker 140 is coupled to a cartridge housing, which is supported by aframe of the flow cytometer 100 at a location along the optical path.When the filter mask 120 is installed in the flow cytometer 100, thefilter mask 120 is positioned so that the origin point 126 is alignedwith an axis A2 (shown in FIG. 2) of the optical path.

The inner edge 154 of the outer blocker 140 defines the outerperipheries of the apertures 124, and therefore defines the maximumradiation angles that may proceed along the optical path. In thisexample, the inner edge 154 is elliptical. Other embodiments have othershapes, such as circular, square, or rectangular. The half height H6 ofthe inner edge 154 defines the maximum vertical radiation angle of lightrays that are permitted to pass through the filter mask 120. The halfwidth W6 of the inner edge 154 defines the maximum horizontal radiationangle of light rays that are permitted to pass through the filter mask120. Typically the filter mask 120 is vertically and horizontallysymmetrical. Asymmetrical filter masks can also be formed in otherembodiments.

The secondary blocker 142, which includes portions 142A and 142B in someembodiments, is arranged at a location between the origin point 126 andthe inner edge 154 of the outer blocker 140, to block a portion of thelight rays that would otherwise pass through the outer blocker 140. Thesecondary blocker 142 can be formed in a linear or a radialconfiguration. The example in FIG. 4 illustrates the linearconfiguration including linear secondary blocker 142. The example inFIG. 5 illustrates the radial configuration, including radial secondaryblocker portions 142C and 142D.

With continued reference to the example shown in FIG. 4, the secondaryblocker 142 has a rectangular shape that extends horizontally betweenthe inner edge 154 of the outer blocker 140. The secondary blockerportion 142A includes an outer edge 156 and an inner edge 158. The outeredge 156 is positioned at a height H7 from the origin point 126 and theinner edge is positioned at a height H8 from the origin point 126. Thesecondary blocker portion 142A extends therebetween, and is configuredto block light rays having radiation angles causing the light rays topass between heights H7 and H8 along the optical path. The thickness ofthe secondary blocker 142 is the difference between height H7 and heightH8, which is less than the height H6 of the inner edge 154. Thesecondary blocker portion 142B typically has the same shape as thesecondary blocker portion 142A, and is arranged at an opposite side ofthe filter mask 120. Apertures 124 positioned above and below thesecondary blocker permit light rays of different radiation angles topass above and below each of the secondary blocker portions 142A and142B. In some embodiments, light rays LR of different radiation angles(both greater than and less than the angles blocked by the secondaryblocker) are permitted to pass through apertures 124 of the filter maskand are collected and analyzed by a single sensor analyzer (as shown inFIG. 1).

The central blocker 144 extends horizontally through the origin point126 and between opposite sides of the inner edge 154 of the outerblocker 140, to block a portion of the light rays that have less than aminimum vertical radiation angle. In this example, the central blocker144 is rectangular having outer edges 160 and 162. The outer edges 160and 162 extend horizontally and are positioned a height H9 away from theorigin point 126. In other embodiments, the edges 160 and 162 arecurved, such as having a arcuate, partial circular, or parabolic shape.

The bullseye blocker 146 is typically centered around the origin point126 and is configured to block light rays having a magnitude of lessthan a minimum radiation angle. In some embodiments the bullseye blocker146 has a circular shape. Other embodiments have other shapes, such aselliptical, square, or rectangular. The bullseye blocker has an outeredge 164. In this example, the outer edge 164 has a half height H10 anda half width W10. When the bullseye blocker has a circular shape, theheight H10 and width W10 are equal to the radius of the circular shape.The bullseye blocker 146 operates to block light rays from the lightbeam that are not radiated by the fluid, as well as those that have aradiation angle of less than the minimum radiation angle. By blockingthis bright portion of the light rays, the signal to noise ratio can besignificantly improved, for example. However, a similar function canalso or alternatively be performed by the central blocker 144 in someembodiments.

FIG. 5 is a front elevational view illustrating another example of afilter mask 120. The filter mask shown in FIG. 5 is generally the sameas that shown in FIG. 4, except that in this example the secondaryblocker 142 has a radial configuration. Accordingly, the descriptions ofthe filter mask 120, body 122, outer blocker 140, central blocker 144,and bullseye blocker 146 are not repeated here.

In this example, the secondary blocker 142 has a radial configurationincluding secondary blocker portions 142C and 142D, and includes anouter edge 156 and an inner edge 158. The secondary blocker 142 extendsbetween the outer edge 156 and the inner edge 158.

In some embodiments, the edges 156 and 158 have a circular shape. Otherembodiments have other shapes, such as elliptical, square, orrectangular. The outer edge 156 has a height H7 and a width W7. Theinner edge 158 has a height H8 and a width W8.

FIGS. 6-9 illustrate several additional examples of filter masks 120.FIGS. 6-7 illustrates a first category of filter masks referred to assmoothness masks. FIGS. 8-9 illustrate a second category of filter masksreferred to as separation masks.

FIG. 6 is a front elevational view of an example filter mask 120A. Theexample filter mask 120A includes a body 122 and apertures 124. The body122 includes an origin point 126, an outer blocker 140, a secondaryblocker 142, and a central blocker 144. Filter mask 120A is a firstexample of a smoothness mask.

As discussed herein, the physical dimensions of the filter mask 120 canbe described in terms of radiation angles of light rays, because theradiation angles correspond to particular physical positions within theoptical path. The radiation angles are proportional to the actualphysical dimensions, which can be computed based on the specificphysical location of the filter mask 120 in the optics system 108.Therefore, FIG. 6 identifies the scale of the example filter mask 120Aaccording to vertical (−50° to +50°) and horizontal (−15° to +15°)radiation angles, and such angles are further discussed below.

The outer blocker 140 has an outer edge 152. In this example, the outeredge is positioned at vertical radiation angles of +/−50° and athorizontal radiation angles of +/−15°.

The outer blocker 140 also has an inner edge 154 having an ellipticalshape. The top and bottom of the inner edge 154 are positioned at thevertical radiation angles of +/−45°, and the sides of the inner edge 154are positioned at the vertical radiation angles of +/−12°.

In this example, the secondary blocker 142 is a linear secondaryblocker. The secondary blocker 142 includes portions 142A and 142B, eachhaving outer edges 156 and inner edges 158. The outer edges 156 arearranged at the vertical radiation angles of +/−39°, and the inner edges158 are arranged at the vertical radiation angles of +/−33°.

The central blocker 144 has outer edges 160 and 162. The outer edges 160and 162 are arranged at the vertical radiation angles of +/−16°.

FIG. 7 is a front elevational view of an example filter mask 120B. Theexample filter mask 120B includes body 122 and apertures 124. The body122 includes an origin point 126, an outer blocker 140, a secondaryblocker 142, and a central blocker 144. Filter mask 120B is a secondexample of a smoothness mask.

The outer blocker 140 has an outer edge 152. In this example, the outeredge is positioned at vertical radiation angles of +/−50° and athorizontal radiation angles of +/−15°.

The outer blocker 140 also has an inner edge 154 having an ellipticalshape. The top and bottom of the inner edge 154 are positioned at thevertical radiation angles of +/−42°, and the sides of the inner edge 154are positioned at the vertical radiation angles of +/−12°.

In this example, the secondary blocker 142 is a radial secondaryblocker. The secondary blocker 142 includes outer edges 156 and inneredges 158. The outer edges 156 are circular having a radius thatintersects with the vertical radiation angles of +/−37°, and the inneredges 158 are circular having a radius that intersects with the verticalradiation angles of +/−31°. In other words, the secondary blocker 142 isarranged and configured to block light rays having radiation anglemagnitudes between 31° and 37°.

The central blocker 144 has outer edges 160 and 162. The outer edges 160and 162 are arranged at the vertical radiation angles of +/−22°.

FIG. 8 is a front elevational view of another example filter mask 120C.The example filter mask 120C includes body 122 and apertures 124. Thebody 122 includes an origin point 126, an outer blocker 140 and acentral blocker 144. Filter mask 120C is a first example of a separationmask.

The outer blocker 140 has an outer edge 152. In this example, the outeredge is positioned at vertical radiation angles of +/−50° and athorizontal radiation angles of +/−15°.

The outer blocker 140 also has an inner edge 154 having an ellipticalshape. The top and bottom of the inner edge 154 are positioned at thevertical radiation angles of +/−19°, and the sides of the inner edge 154are positioned at the vertical radiation angles of +/−12°.

The central blocker 144 has outer edges 160 and 162. The outer edges 160and 162 are arranged at the vertical radiation angles of +/−12.2°.

FIG. 9 is a front elevational view of an example filter mask 120D. Theexample filter mask 120D includes body 122 and apertures 124. The body122 includes an origin point 126, an outer blocker 140, a secondaryblocker 142, and a central blocker 144. Filter mask 120D is a secondexample of a separation mask.

The outer blocker 140 has an outer edge 152. In this example, the outeredge is positioned at vertical radiation angles of +/−50° and athorizontal radiation angles of +/−15°.

The outer blocker 140 also has an inner edge 154 having an ellipticalshape. The top and bottom of the inner edge 154 are positioned at thevertical radiation angles of +/−23°, and the sides of the inner edge 154are positioned at the vertical radiation angles of +/−12°.

In this example, the secondary blocker 142 is a linear secondaryblocker. The secondary blocker 142 includes portions 142A and 142B, eachhaving outer edges 156 and inner edges 158. The outer edges 156 arearranged at the vertical radiation angles of +/−17°, and the inner edges158 are arranged at the vertical radiation angles of +/−15°.

The central blocker 144 has outer edges 160 and 162. The outer edges 160and 162 are arranged at the vertical radiation angles of +/−12.2°.

FIGS. 10-11 illustrate another example of the optics system 108, shownin FIG. 1. FIG. 10 is a cross-sectional side view of the example opticssystem 108. FIG. 11 is a cross-sectional top view of the example opticssystem 108.

In this example, the optics system 108 includes a collection opticsassembly 161, re-imager 163, collimator 165, beam separating assembly167, filter masks 120, as well as additional possible optical components169.

Although this example is illustrated and described with reference to aparticular physical implementation of the optics system 108, such asincluding particular types of lenses and particular lens configurations,other embodiments can have other configurations. Additional examples ofpossible optics assemblies are illustrated and described in more detailin U.S. Patent Application Ser. No. 61/793,771, titled OPTICS SYSTEM FORA FLOW CYTOMETER, and filed on even date herewith, the disclosure ofwhich is hereby incorporated by reference in its entirety.

In some embodiments the collection optics assembly 161 includes lens 172and triplet 174, a re-imager 163 including doublets 176 and 178, and acollimator 165 including a doublet 180.

Additionally, some embodiments include a beam separating assembly 167that is arranged and configured to separate the light rays into two ormore separate beams, such as a beam 192 and a beam 194 (FIG. 11). Inthis example, the beam separating assembly 167 includes a beam splitter202 and a mirror 204. The beam splitter 202 is positioned in the opticalpath of the optics system 108 and is configured such that half of thelight rays are reflected toward the mirror 204, forming the beam 194(FIG. 11), and the other half of the light rays are transmitted, formingthe beam 192 (FIG. 11). In some embodiments, the mirror 204 is arrangedto redirect the beam 194 toward the sensor analyzer 110, so that thebeams 192 and 194 are parallel. The light rays LR can be separated intoadditional beams using additional beam splitters, if desired. In anotherpossible embodiment, the mirror 204 can be omitted, such that the beam194 continues in a direction perpendicular to beam 192, and anothersensor analyzer 110 (or another portion of the sensor analyzer 110) maybe positioned along the beam 194 path.

Each of the separate beams 192 and 194 can then be separately filteredand analyzed. In this example, each of the beams 192 and 194 is passedthrough a separate filter mask 120 ¹ and mask 120 ². The filter masks120 ¹ and 120 ² can be the same, or they can be different. For example,the filter mask 120 ¹ can be used to permit a selected portion of lightrays to pass that are associated with certain radiation angles θ, andthe filter mask 120 ² can be used to permit another selected portion oflight rays to pass that are associated with other radiation angles θ. Inthis way the sensor analyzer 110 can evaluate multiple portions of theradiated light rays separately and simultaneously for the same portionof the fluid stream F.

In one example embodiment, the filter mask 120 ¹ is a smoothness maskand the filter mask 120 ² is a separation mask. Examples of smoothnessmasks are illustrated in FIGS. 6-7, and examples of separation masks areillustrated in FIGS. 8-9. Smoothness and separation masks are alsodiscussed in further detail with reference to FIGS. 12-13.

Some embodiments include one or more additional optical components 169.Examples of the additional optical components include filter components206, lenses 208, and aperture components 210.

The filter components 206 are provided in some embodiments to furtherfilter the light rays before they are passed to the sensor analyzer.Examples of filter components 206 include spectral filters, neutraldensity filters, and polarizing filters.

The lenses 208A and 208B are provided to converge the light rays to afocal point to pass the light rays through aperture components 210A and210B. The aperture components 210A and 210B are positioned at the focalpoints of the lenses 208A and 208B and are configured to block straylight from the sensor analyzer. Additional aperture components 210 cansimilarly be included at other focal points.

In the example shown in FIG. 11, the overall path lengths of beams 192and 194 are not equal. More specifically, because the beam 194 is offsetby the beam splitter a distance equal to the distance between the beamsplitter 202 and the mirror 204, the beam 194 travels a longer distancethan the beam 192. In other possible embodiments, the optics system 108is adjusted to provide equal path lengths. For example, the offset ofbeam 194 can be accounted for by either increasing the path length ofbeam 192 by an equal distance, or by decreasing the path length of beam194 by an equal distance. As a more specific example, the distancebetween the filter components 206 and the lenses 208 (e.g., in thecollimated or pseudo-collimated region), can be increased or decreasedto provide beams 192 and 194 of equal path lengths. For instance, insome embodiments the distance between beam splitter 202 and the sensoranalyzer 110 is increased (between filter components 206A and lens 208A)a distance equal to the distance between beam splitter 202 and mirror204, so that the path lengths for beams 192 and 194 are equal.Alternatively, in another possible embodiment the distance betweenmirror 204 and sensor analyzer 110 can be reduced by the same distance.

FIG. 12 is a schematic diagram illustrating an exemplary responseprofile for a smoothness mask. Some specific examples of smoothnessmasks are illustrated and described with reference to FIGS. 6-7.Response profiles for three different types of particles (Types A, B,and C) are illustrated. The specific data illustrated in FIG. 12 ishypothetical but provided to help illustrate the concepts discussedbelow.

A smoothness mask is a filter mask that, for a given type of particle,exhibits a linear, or substantially linear, response to variations inthe size of particles contained within the sample. As a result, when theparticle type is known, the size of the particle can be determined veryprecisely. FIG. 12 depicts an example of such a response. In thisexample, the radiation intensity increases linearly as a function of theparticle size, for a given type of particle. In other words, largerparticles of a particular type (e.g., Type A) result in a greaterdetected radiation intensity than smaller particles of the same type.Therefore, when a sample contains a single known type of particle, or asingle set of particles having a common response profile, the detectedradiation intensity provides a direct indication of the sizes of theparticles.

A smoothness mask may be somewhat sensitive to variations in the typesof particles present in a sample. For example, when a sample containsmultiple different types of particles (e.g., Types A, B, and C), thefilter mask may not exhibit a linear response across the multipledifferent types of particles. As one example, if the sensor analyzer 110(FIG. 1) detects a radiation intensity for a given particle of 4×10⁷,and multiple different types of particles (e.g., Types A, B, and C) arepresent in the sample, it is difficult for the cytometer 100 todetermine the particle size based solely on the detected radiationintensity. As shown, the particle size may be 20 μm for particle Type A,27 μm for particle Type B, or 33 μm for particle type C. As a result, asmoothness mask may cause the detected radiation intensity for a smallerparticle of a first type (e.g., a 20 μm particle of Type A) to begreater than the radiation intensity detected for a larger particle of asecond type (e.g., a 30 μm particle of Type C). In this scenario, it canbe difficult to determine the relative or actual sizes of the particlesbased solely on the radiation intensity detected when using a singlesmoothness mask.

In some embodiments, the smoothness mask is selected to minimize ripple,resulting in a reduced path length for the scatter intensity function.The minimum path length possible is a straight line, such as illustratedin FIG. 12.

FIG. 13 is a schematic diagram illustrating an exemplary responseprofile for a separation mask. Some specific examples of separationmasks are illustrated and described with reference to FIGS. 8-9.Response profiles for three different types of particles (Types A, B,and C) are illustrated. The specific data illustrated in FIG. 13 ishypothetical but provided to help illustrate the concepts discussedbelow.

A separation mask is a filter mask in which the radiation intensitiesdetected for given particles sizes have as little variation as possibleacross multiple different types of particles. In a separation mask,reduced variation is more important than a linear response, andtherefore the separation mask may have more ripple (i.e., greater pathlength) than a smoothness mask.

In the example shown in FIG. 13, the response profiles obtained with theseparation mask are not perfectly linear. But, the response profiles formultiple different particle types (e.g., Types A, B, and C) are veryclose together. As a result, any of the particles (Types A, B, or C)having a size of 20 μm result in a detected radiation intensity of about4.5×10⁷, and particles having a size of 30 μm result in a detectedradiation intensity of about 6×10⁷. The separation mask is selected tomaintain as much separation (“S” in FIG. 13) as possible in theradiation intensities detected for particles having different sizes,regardless of the type of the particle. For example, the separation S isdifference in the radiation intensity detected for a particle of a givensize (e.g., 20 μm) of type A, and the radiation intensity detected for aparticle of a larger given size (e.g., 30 μm) of type C. If theseradiation intensities overlap, then the separation of the filter mask isnot adequate, because the cytometer may not be able to differentiatebetween the particles of different sizes. When they do not overlap,however, as shown in FIG. 13, the cytometer can distinguish between thetwo different sized particles, even though they are different types ofparticles. In fact, particles that are even closer in size could bedistinguished from each other using the separation mask example shown inFIG. 12.

Because of the separation in the response profile provided by aseparation mask, the separation mask can be used to determinedifferences in particle sizes even when particles of different types arepresent in the sample.

In some cases, even better results can be obtained by utilizing both asmoothness mask and a separation mask simultaneously in the cytometer toevaluate the particles within a sample. By separating the beam into twoor more separate beams, such as shown in FIGS. 10-11, a smoothness maskcan be inserted as one of the filter masks (e.g., 120 ¹) and aseparation mask can be inserted as another of the filter masks (e.g.,120 ²), to independently and simultaneously evaluate light raysassociated with the same particle. For example, the radiation intensitydetermined using the separation mask can be used to determine theapproximate size of the particle, regardless of the particle type. Usingthis information, the flow cytometer can then use the radiationintensity determined using the smoothness mask to determine the particletype and a more precise size of the particle. The results obtained byusing two or more different filter masks simultaneously, can thereforegenerate better results than using either of the two or more filtermasks by themselves.

Additional types of filters can also be used to obtain even moreinformation regarding a sample. The filters can include one or moreadditional filter masks that selectively block or pass light rays basedon radiation angle, or can include one or more spectral filters, neutraldensity filters, and polarizing filters.

FIGS. 14-16 illustrate an example mask holder 252, including a housing254 and a filter mask 120. FIG. 14 is a front perspective view, FIG. 15is a rear perspective view, and FIG. 16 is a front elevation view. Inthis example, the housing 254 includes a top 260, sides 262 and 264, andbottom 266. Also illustrated in this example are engagement feature 272,and grip features 274 and 276.

In this example, the housing 254 includes a top 260, sides 262 and 264,and bottom 266. In some embodiments, the flow cytometer 100 (FIG. 1) isconfigured to receive removable filter masks 120. In this example, thefilter masks 120 are contained in mask holders 252 that can be easilyinserted into or removed from the flow cytometer 100 as desired. Aplurality of different mask holders 252 having different filter masksare provided in some embodiments, and the particular mask holder can beselected by the operator depending on the type of sample, or the type ofevaluation to be performed, for example.

In some embodiments, the mask holder 252 is formed of a single piece ofmaterial. The mask holder 252 can be molded for example. In anotherpossible embodiment, the mask holder 252 is formed of a solid piece ofmaterial which is then machined, etched, or otherwise formed into thedesired configuration. In other embodiments, the mask holder 252 isformed of two or more pieces of material. Examples of possible materialsinclude plastic, metal, glass, and combinations of these or othermaterials. One or more coatings can also be applied, such as a paint. Insome embodiments the materials and/or coatings are light absorbentand/or non-reflective.

The housing 254 is sized and shaped, in some embodiments, for insertionwithin a correspondingly sized receptacle aligned at the appropriatelocation within the optics system of the cytometer.

In some embodiments, the top 260 includes an engagement feature 272. Acover or latch over the cytometer's receptacle is configured to engagewith the engagement feature 272 when the mask holder 252 is properlyinserted into the receptacle.

In some embodiments, sides 262 and 264 include grip features 274 and276. In this example, the grip features 274 and 276 are configured to begrasped by tips of the operator's fingers to permit easier removal ofthe mask holder 252 from the cytometer's receptacle.

In some embodiments, the mask holder 252 has a height H11, a length L11,and a width W11 as shown in FIGS. 14-16. Various embodiments can havevarious sizes. As one example, the height H11 and the length L11 are ina range from about 0.5 inches to about 2 inches. Some embodiments have aheight H11 and a length L11 of about 1 inch. As another example, thewidth W11 is in a range from about 0.1 inches to about 0.5 inches. Someembodiments have a width W11 of about 0.25 inches.

In some embodiments a thickness of the filter mask 120 is less than thewidth W11 of the mask holder 252. As one example, the thickness is in arange from about 10 thou to about 30 thou. Some embodiments have athickness of about 20 thou (0.5 mm).

The mask holder 252 illustrated herein is provided by way of example,but a wide variety of alternative configurations are also possible.

As described herein, some embodiments of the flow cytometer 100 includeone or more types of computer readable media. Computer readable mediaincludes any available media that can be accessed by the computingdevice 112. By way of example, computer readable media include computerreadable storage media and computer readable communication media.

Computer readable storage media includes volatile and nonvolatile,removable and non-removable media implemented in any device configuredto store information such as computer readable instructions, datastructures, program modules or other data. Computer readable storagemedia includes, but is not limited to, random access memory, read onlymemory, electrically erasable programmable read only memory, flashmemory or other memory technology, compact disc read only memory,digital versatile disks or other optical storage, magnetic cassettes,magnetic tape, magnetic disk storage or other magnetic storage devices,or any other medium that can be used to store the desired informationand that can be accessed by the computing device 112. Computer readablestorage media does not include computer readable communication media.

Computer readable communication media typically embodies computerreadable instructions, data structures, program modules or other data ina modulated data signal such as a carrier wave or other transportmechanism and includes any information delivery media. The term“modulated data signal” refers to a signal that has one or more of itscharacteristics set or changed in such a manner as to encode informationin the signal. By way of example, computer readable communication mediaincludes wired media such as a wired network or direct-wired connection,and wireless media such as acoustic, radio frequency, infrared, andother wireless media. Combinations of any of the above are also includedwithin the scope of computer readable media.

In some embodiments, the term “substantially” refers to a deviation ofless than 5%. In other embodiments, the term refers to a deviation ofless than 1%. Yet other embodiments have a deviation of less than 0.1%.Other embodiments have other magnitudes of deviation.

The various embodiments described above are provided by way ofillustration only and should not be construed to limit the claimsattached hereto. Those skilled in the art will readily recognize variousmodifications and changes that may be made without following the exampleembodiments and applications illustrated and described herein, andwithout departing from the true spirit and scope of the followingclaims.

What is claimed is:
 1. A filter mask for use in a flow cytometer, thefilter mask comprising: a body including: light blocking featuresincluding: an outer blocker extending between an outer edge and an inneredge, the inner edge being positioned around an origin point; a centralblocker extending through the origin point and between opposite sides ofthe inner edge of the outer blocker; a bullseye blocker centered aroundthe origin point and overlapping the central blocker; and a secondaryblocker extending between the opposite sides of the inner edge of theouter blocker at a distance away from the origin point, the secondaryblocker configured to block a portion of light rays having radiationangles between maximum and minimum radiation angles; and light passingapertures defined by spaces between the inner edge of the outer blocker,the central blocker, and the secondary blocker, and configured to permitlight rays to pass through the body.
 2. The filter mask of claim 1,wherein the outer blocker is arranged and configured to block light rayswithin the flow cytometer associated with radiation angles greater thanmaximum vertical and horizontal radiation angles.
 3. The filter mask ofclaim 1, wherein the secondary blocker is arranged and configured toblock at least some light rays within the flow cytometer associated withradiation angles less than the maximum vertical and horizontal radiationangles, and is positioned between the outer blocker and an origin pointof the filter mask.
 4. The filter mask of claim 3, wherein the secondaryblocker has a rectangular shape.
 5. The filter mask of claim 3, whereinthe secondary blocker has a curved shape.
 6. The filter mask of claim 1,wherein the filter mask is contained in a mask holder.
 7. The filtermask of claim 1, wherein the filter mask is a smoothness filter having asubstantially linear response profile for a single type of particle. 8.The filter mask of claim 1, wherein the filter mask is a separationfilter having a separation in the response profiles of particles ofdifferent types.
 9. A flow cytometer comprising: a flow nozzleconfigured to provide a fluid along a flow path, the fluid includingsample particles therein; a light source configured to generate a lightbeam directed toward the flow path, wherein when the light beamintersects with the flow path, light rays are radiated by the fluid andthe particles at radiation angles; an optics system configured toreceive the radiated light rays and to direct the light rays along anoptical path, the optics assembly including at least a first filtermask, wherein the first filter mask is the filter mask of claim 1; and asensor analyzer arranged at an end of the optical path to collect andanalyze light rays passing through the optics system.
 10. The flowcytometer of claim 9, wherein the optics system comprises a receptacle,and wherein the first filter mask is removable from the receptacle. 11.The flow cytometer of claim 9, wherein the optics system furthercomprises: a beam separating assembly arranged along the optical path toseparate the light rays into at least two separate beams, wherein thefirst filter mask is arranged along a first of the separate beams; and asecond filter mask arranged along a second of the separate beams. 12.The flow cytometer of claim 11, wherein the first filter mask passeslight rays having a first set of radiation angles, and wherein thesecond filter mask passes light rays having a second set of radiationangles.
 13. The flow cytometer of claim 12, wherein at least some of theradiation angles are the same in the first set and the second set.
 14. Amethod of evaluating a particle with a flow cytometer, the methodcomprising: passing a particle in a fluid along a fluid flow path;illuminating the particle and the fluid with a light beam; collectinglight rays radiated from the fluid and the light beam with an opticssystem; selectively blocking some of the light rays having certainradiation angles with a first filter mask, wherein the first filter maskis the filter mask of claim 1; selectively passing some of the lightrays with the first filter mask; and detecting light rays passed by thefirst filter mask with a sensor analyzer to evaluate at least onecharacteristic of the particle.
 15. The method of claim 14, wherein theat least one characteristic is selected from a size of the particle anda type of the particle.
 16. The method of claim 14, further comprisingseparating the light beam into at least two separate beams with theoptics system.
 17. The method of claim 16, wherein the selectivelyblocking and the selectively passing involves a first set of separatebeams using the first the filter mask, and further comprising:selectively blocking some of the light rays of a second set of separatebeams having certain radiation angles using a second filter mask,wherein the radiation angles blocked with the second filter mask aredifferent than the radiation angles blocked with the first filter mask.18. The method of claim 14, further comprising: receiving the firstfilter mask into a receptacle of the flow cytometer, the first filtermask being housed in a mask holder, wherein receiving the first filtermask into a receptacle of the flow cytometer occurs before passing theparticle in the fluid along the fluid flow path.
 19. The filter mask ofclaim 1, wherein the secondary blocker extends in a horizontal directionat a distance above the origin point.
 20. The filter mask of claim 1,wherein the secondary blocker extends in a horizontal direction at adistance below the origin point.
 21. The filter mask of claim 1, whereinthe secondary blocker comprises first and second portions, each portionpositioned at an opposite side of the origin point.
 22. The filter maskof claim 1, wherein the central blocker is configured for blocking lightrays having radiation angles less than a minimum vertical radiationangle.
 23. The filter mask of claim 1, wherein the bullseye blocker isconfigured to block light rays that are not radiated and that have aradiation angle less than the minimum radiation angle.
 24. The filtermask of claim 1, wherein the central blocker has a rectangular shape andthe bullseye blocker has a rounded shape.
 25. The filter mask of claim1, wherein the central blocker has a first outer edge defining a firsthalf distance from the origin point, wherein the bullseye blocker has asecond outer edge defining a second half distance from the origin point,and wherein the second half distance is larger than the first halfdistance.